Metastasis, the dissemination of malignant cells from the primary tumor to secondary sites, is a highly complex, multi-step process. In order to understand mechanistically the process of metastasis it will be necessary to identify those molecules and pathways that specifically contribute to tumor dissemination and secondary site growth. To help achieve our goal of identifying functionally relevant molecules in metastasis, we have used subtractive immunization to generate unique function-blocking monoclonal antibodies (mAbs) raised against the highly aggressive human tumor HEp3. The mAbs are screened in vivo as inhibitors of the metastatic cascade first in a recently-modified chick embryo assay and are then confirmed in the SCID mouse metastasis model. Utilizing the subtractive-generated mAbs and analytical proteomics technology, two membrane-anchored protein antigens that contribute to tumor progression in our metastasis models have been identified. One antigen is a new type I transmembrane glycoprotein SIMA-135/CDCP-1 (SIMA-135), and the other is a known member of the tetraspanin family, PETA3/CD151 (PETA3). The Specific Aims are: (1). To elucidate the functional role of the new antigen, SIMA-135, in human tumor metastasis. Expression of the SIMA-135 gene will be downregulated in metastatic cells, upregulated in non-aggressive cells and the resulting cell isolates will be analyzed for in vivo alterations in behavior. A search for potential SIMA-135 partner molecules also will be conducted. (2). To demonstrate the mechanism and role of the tetraspanin, PETA3, in the metastatic process. The position where PETA3 functions in the metastatic cascade will be elucidated and an in vivo structure-function analysis of PETA3 will be carried out. (3). To correlate the expression, appearance, and distribution of SIMA-135 and PETA3 antigens with clinical aspects of human malignant disease. (4). To extend the utilization of subtractive-immunization and function-blocking mAbs by applying these in vivo experimental approaches to other human tumor cell lines manifesting distinct malignant phenotypes. The overall goal is to identify protein antigens that function directly in the metastatic process. Such antigens could serve as potential diagnostic markers or therapeutic targets for human cancer.